Toll-like receptors (TLRs) and interferons (IFNs) induce genes that mediate innate immunity, and shape the adaptive immune response. A subset of IFN-stimulated genes (ISGs) is induced by the TLR agonists doublestranded RNA (dsRNA) and lipopolysaccharide (LPS), suggesting that they function in host defense. ISG15 stands out among these ISGs as a protein with immunomodulatory properties that is one of the earliest and strongest responses to these stimuli; the specific function(s) of ISG15 in innate immunity are not known. ISG15 is an ubiquitin-like protein that acts within cells to posttranslationally modify cellular proteins. Free, unconjugated ISG15 is released from cells where it exhibits cytokine-like activities. Virus, IFN, and TLR agonists induce comparable levels of ISG15. However, predominantly free ISG15 is observed in virus and TLR agonist-stimulated cells, leading to its enhanced secretion; whereas, IFN treatment results in a robust conjugation of ISG15 to cellular proteins, favoring its retention in cells. We hypothesize that ISG15 serves a bifunctional role in innate immunity: i) as an extracellular cytokine that is induced by virus, bacteria, and TLR agonists; and ii) as an intracellular mediator of IFN action through its conjugation to cellular proteins. Thus, the differential regulation of ISG15 conjugating and deconjugating enzymes by distinct inducers dictates the extent of ISG15 conjugation/secretion, and hence its predominant biologic function. To understand the functional significance of ISG15 conjugation, it is essential to identify ISG15 conjugates. Proteasome inhibition dramatically increases the levels of specific ISG15 conjugates; we will employ this condition to purify ISG15 conjugates (aim 1). Mapping and mutagenesis of ISG15 conjugation sites will determine how conjugation influences protein stability and subcellular location (aim 2). To dissect the mechanism by which distinct stimuli differentially regulate ISG15 conjugation and secretion, the induction of ISG15 conjugating and deconjugating enzymes by virus, IFN, dsRNA and LPS will be determined (aim 3). To place ISG15 induction in the context of the innate immune response, we will measure the expression of intracellular and extracellular ISG15 in response to microbial stimuli in mice (aim 4). An understanding of how ISG15 functions in the cytokine network may reveal strategies to manipulate immune function for therapeutic applications.